Expression of mammalian genes in several plants including tobacco and Arabidopsis has been recognized as efficient, low cost, non-sterile bioreactors for the production of proteins valuable to both medicine and industry (Ma and Hein 1995). Recently, evidence was presented that demonstrated that the four chains of the secretory immunoglobulin were properly expressed and assembled in plants and that the antibody was fully functional (Ma et al 1995). Furthermore, bacterial (Haq et al 1995), and viral (Mason et al 1996) antigens produced in transgenic tobacco and potato effectively immunized mice when the transgenic potato was administered orally. Oral administration of protein antigens can result in a diminished immune response to subsequent systemic administration (eg., by injection) of the same antigen, a process known as oral tolerance (Mowat, 1987). Oral tolerance has recently been explored as a potential antigen-specific immunotherapeutic strategy for the treatment of organ-specific autoimmune diseases. In animal models, oral administration of disease inducing autoantigens has been shown to inhibit several experimental autoimmune diseases, including animal NOD diabetes, uveoretinitis, experimental autoimmune encephalomyelitis (EAE), and rheumatoid arthritis (RA). However, one limitation with clinical use of the oral tolerance approach is the cost of protein production, as oral tolerance requires the ingestion of relatively large amounts of protein antigens, often in excess of quantities that can be provided by conventional protein synthesis or fermentation systems.
As the immune system is specific in the recognition of protein antigens, it is preferred that the antigens are mammalian and species specific to elicit the desired reduction in antigen directed immune responses. The use of plants as an expression system for the production of mammalian antigen proteins offers unique advantages beyond high production yields at competitive low cost. These advantages include reduced health risks from possible pathogen contamination as would be the case with extraction from mammalian tissues, correct modification and assembly of protein leading to function of proteins expressed by the plant when required or desired, optimization of high expression levels, and the ability to induce oral immune tolerance without extensive purification of the plant material. Using edible plant tissue leads to major cost reductions due to the simplicity of the delivery system.
Peripheral immune responses can be downregulated with oral administration of protein antigens, and this effect is antigen and protein specific. Human volunteers fed keyhole limpet hemocyanin (KLH) demonstrated reduced peripheral immune responses to KLH on recall responses of lymphocytes re-exposed to that antigen in vitro (Matsui, M. et al., 1996, Ann NY Acad Sci 778:398-404). Therefore, oral tolerance can be demonstrated in humans. The magnitude of response may be influenced by several factors, including expression levels of the transgenic protein of interest within plants, or the presence of immune adjuvants or response modifiers present within the composition.
Plants also may represent an ideal expression system for oral immune tolerance for several additional factors. Firstly, immune tolerance requires activation events within T lymphocytes, and plants by their composition which includes lectins may facilitate this. Additionally, the accumulation of transgenic proteins within plant cell compartments may protect the antigen from degradation in the digestive tract. This could augment oral immune tolerance by delivering transgenic plant protein to effector sites in the gut distal to the digestive environment of the stomach and duodenum (e.g. Kong et al, 2001, P.N.A.S, 98:11539-11544).
The use of plants as an expression system for production of mammalian antigenic proteins offers several unique advantages, including high production yields at competitive low cost, reduced health risks from pathogen contamination, and correct modification and assembly of foreign proteins. An additional advantage of a plant production system is that proteins, in the case of oral immune tolerance induction, may be used directly without extensive purification resulting in further cost reductions. In this regard it has been noted that proteins produced in transgenic tobacco required purification prior to administration. Clearly steps involving purification or processing are undesirable if ease of oral administration is to be maximized as well as minimizing any associated costs for production.
Numerous plants have proven themselves to be amenable to transformation with heterologous genes and for some time tobacco has been the model system for plant transformation. Despite the fact that crop-protection focused biotechnologies have not found application in non-food crop plant production, a major role does remain for such plants as bioreactors. An example of a non-food crop plant is tobacco, which is capable of producing high levels of soluble protein (fraction 1 protein, F1P; Woodleif et al 1981) and pilot systems have been developed to purify this fraction for use as a high protein dietary supplement (Montanari et al 1993).
From both a regulatory and public safety stand point non-food crop plants are ideal species for the transgenic production of biologically active proteins. Non-food crop plants minimize the risk of accidental leakage of transgenic plant material expressing genes for biologically active proteins into the human food chain. Other plant bioreactor systems based on canola (Rooijen et al. 1995), potato (Manson et al 1996), rice and cassava (Ma and Hein 1995) do not offer this advantage. Furthermore, non-food crop plants can be selected so that production in areas where there are no naturally occurring wild species further minimizes the risk of gene leakage to the local flora, an example of this would be to grow tobacco in regions where tobacco does not overwinter, such as Canada. With any non-food crop plant, transgenic proteins can be produced using any tissue or organ of the plant. However if protein production is based on leaves, not seeds or tubers, and when coupled with the fact that the leaves are harvested before flowering there is virtually no risk of uncontrolled bioreactor plants occurring in future crop seasons.
However, many non-food crop plants contain high levels of secondary plant products making plant tissue obtained from these plants unsuitable for direct oral administration. Earlier studies have described the administration of tobacco-derived proteins to mice, however, the proteins were in a partially purified form. For example the study by Mason et al (1996) involved the direct oral administration of viral antigens expressed in potato tuber and tobacco. The potato tuber samples were directly fed to mice, yet the antigen, when obtained from tobacco, had to be partially purified using sucrose gradients prior to administration to mice.
The breeding of low alkaloid-containing tobacco plants has been reported (Chaplin 1977), however, use of such a plant as a bioreactor has not been suggested. Recently, specific alteration of nicotine levels within tobacco, by either over expression (i.e. increasing) or antisense expression (decreasing) of putrescine N-methyltransferase, a rate limiting enzyme involved in the nicotine biosynthetic pathway, has been suggested (U.S. Pat. No. 5,260,205, issued Nov. 9, 1993 and U.S. Pat. No. 5,369,023, issued Nov. 29, 1994; inventors Nakatani and Malik). However, these methods are directed to the alteration of nicotine levels so that the levels of other alkaloids that affect the flavours and aroma of tobacco are not modified in any manner. These plants are not ideal for use as a non-food crop plant as describe herein, since the levels of only a select group of alkaloids have been reduced. Further, no nicotine free plants were actually produced, nor was the use of these transgenically modified tobacco plants, as a bioreactor for the synthesis of proteins of interest, suggested. However, such methods may be used and augmented in order to produce non-food crop plants where the total alkaloid level is reduced. Such an approach may be useful for the production of low alkaloid plants as a bioreactor for the synthesis of proteins of interest as contemplated by this invention.
U.S. Pat. No. 6,338,850 and EP 720, 484 discloses the expression of glutamic acid decarboxylase within a plant.
Oral tolerance has been explored as a potential antigen specific immunotherapeutic strategy for the treatment of organ specific autoimmune disease and prevention of transplantation and rejection. However, there are several potential limitations in the success of this approach in human disease, including selection of the appropriate antigen that is specific to disease, intervention in the disease process prior to onset of marked epitope spreading in which the targeting of the initial peptide is extended to associated peptides within the triggering protein, intervention in the disease process prior to onset of advanced clinical symptoms, and the requirement for large amounts of protein antigen suitable for clinical use. This latter issue leads to large costs of protein production from the use of conventional expression systems including transgenic expression in tissue culture and fermentation systems, to produce the amounts of protein needed. Additionally, expression in bacterial systems incapable of glycosylation and correct folding of the protein, leads to the formation of inclusion bodies and marked reduction in levels of recoverable usable protein antigens.
There is a need in the art for a method of making proteins in large quantities and at low cost. Further there is a need in the art for making biologically active proteins in large quantities and low cost, that do not require purification or further processing prior to being administered to a subject. Furthermore, there is a need for enhancing the action of an autoantigen, for example by co-administration of the autoantigen with a cytokin.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.